Gp63 is a highly conserved surface protease of the Leishmania sp. protozoa that participates in several critical steps of the parasite life cycle. The protein varies in abundance at different times in parasite growth, indicating its expression is tightly regulated. During the initial finding period of this grant we found that Leishmania chagasi gp63s are encoded by a family of more than 18 tandemly repeated genes, which we named msp for major surface protease. The cluster contains 3 different classes of msp genes and unrelated genes called mig (msp intervening genes). msp class RNAs are differentially expressed during the parasite growth and life cycle, as are several of the encoded gp63 proteins. The msp/mig cluster of L. chagasi is now one of the best characterized multi-gene clusters of Leishmania sp. The expression of different msp genes is regulated primarily by post- transcriptional mechanisms. Our preliminary data show that sequences responsible for post-transcriptional regulation lie in the 3 'UTR plus downstream intergenic regions of some of these genes. However, we also found that msp/mig transcription is directional and that a non-transcribed region occurs upstream of the entire cluster. Thus, some sequences appear to regulate transcription of the entire msp/mig cluster and other sequences determine the differential expression of individual genes within the cluster. The specific aims of this project are: (l) To study sequences regulating the steady state amounts of the different msp and mig RNAs. (2) To measure the steady state amount, and the rates of translation and degradation of proteins encoded in the msp/mig cluster. (3) To characterize transcription of the msp/mig gene cluster. (4) To study the effects of environmental exposures and life stage changes on the expression of msp and mig genes. The studies are intended to determine the mechanisms through which Leishmania express gp63, a critical virulence-associated protein. They will also provide a model for differential expression of tandemly linked genes in this group of protozoa.